Overview: Project Summary/Abstract The long-term goal of the Genomics of Acute Myeloid Leukemia Program Project Grant (GAML PPG) is to define the genetic and epigenetic events that drive AML progression, relapse, and response to therapy, and to use this information to improve risk assessment and treatment. In the past five years, we have defined the roles of subclonal heterogeneity and evolution for AML progression and relapse, but the essential molecular events that are responsible have not been fully defined by exome and/or bulk RNA- sequencing. We will therefore exploit single cell transcriptomics to more fully define the epigenetic features of clonal evolution (Projects 1 and 3). We have also learned that hematopoietic stem/progenitor cells (HSPCs) that contain functional TP53 mutations are often selected for by chemotherapy and other stressors; the cell- intrinsic and extrinsic processes that contribute to the progression of clonal hematopoiesis to AML will be defined in Project 4. Project 2 will use genomic approaches to define minor histocompatibility antigens that are expressed specifically in AML cells to improve the immunologic therapy of AML. With these studies, we hope to define new targets and novel therapies for high-risk AML patients, via the following projects and cores: Project 1 (Ley): Genomics of Intermediate-Risk AML Progression and Relapse. This project will define the genetic and epigenetic features of intermediate-risk AML with post-chemo relapse, and determine whether the initiating DNMT3AR882H mutation represents a therapeutic target for fully transformed AML cells. Project 2 (DiPersio): Genomics of Allogeneic Transplantation. Genomic approaches will be used to define minor histocompatibility antigens that contribute to the Graft vs. Leukemia effect of allotransplantation; a mouse model of human AML (Dnmt3aR878H + FLT3-ITD) will be used to better define allo-resistance. Project 3 (Walter): Genomics of Secondary AML Progression. This project will define the genetic and epigenetic characteristics of subclones in MDS patients that contribute to progression to secondary AML, and model MDS disease progression in mice using well-defined MDS-initiating mutations (e.g. U2AF1). Project 4 (Link): Genomics of TP53-mutant AML. This project will use genomic approaches to define genetic and epigenetic events that contribute to the initiation and progression of AML initiated by TP53 mutations, and use mouse models to define extrinsic stressors that may contribute to AML progression. Core A (Westervelt). Clinical Database: provides patient enrollment, sample collection, clinical annotation, database management, and statistical support for all projects. Core B (Payton). Specimen Database: provides sample annotation, storage, and preparation for all projects. Core C (Miller). Sequencing and Analysis: provides all data production (scRNA-seq, eWGS, error-corrected sequencing, and custom studies) and comprehensive integrative analyses for all projects. Core D (Ley). Administration: provides administrative support for projects, cores, and the EAB.